This invention relates to the inhibition of xcex1- and xcex3-thrombin-induced cell activation.
Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, SEQ ID NO: 40) is a vasoactive peptide released from the precursor plasma kininogens by plasma and tissue kallikreins and other enzymes (Silva et al., Amer. J. Physiol. 156: 26-274 (1949)). The parent proteins of bradykinin, high (HK) and low (LK) molecular weight. kininogens were recognized to have the ability to inhibit xcex1- and xcex3-thrombin-induced platelet activation (Meloni et al., J. Biol. Chem. 266, 6786 (1991); Puri et al., Blood 77, 500 (1991)). Both low and high molecular weight kininogens have identical amino acid sequences from their amino-terminus through 12 amino acids beyond the carboxy-terminus of bradykinin. Both LK and HK share a common heavy chain (62 kDa), the bradykinin (BK) moiety (0.9 kDa), and the first 12 amino acids of the amino terminal portion of each of their xe2x80x9clight chainsxe2x80x9d (Takagaki et al., J. Biol. Chem. 260, 8601-8609 (1985); Kitamura et al., J. Biol. Chem., 260, 8610-8617 (1985)). This identity corresponds to residues 1 through about residue 383. See Salveson et al., Biochem J. 243, 429 (1986); Kellerman et al., Eur. J. Biochem. 154, 471 (1986). The HK and LK kininogens diverge in the size of their light chains; the light chain of LK is 4 kDa; that of HK is 56 kDa. (Takagaki et al., supra; Kitamura et al., supra.). The kininogens prevent thrombin-induced platelet activation without inhibiting thrombin""s ability to hydrolyze a chromogenic substrate. Full-length kininogens prevent thrombin from binding to platelets. They do not interfere with thrombin""s ability to proteolyze, i.e. cleave fibrinogen, which allows released fibrin monomer to make a fibrin clot. Thus, the prior art indicated that the kininogens"" ability to inhibit thrombin activation of platelets was not due to their direct interaction with the thrombin molecule itself (Meloni et al., supra; Puri et al., supra).
The thrombin inhibitory activity of the kininogens appeared to be localized to an isolated domain 3 of the kininogens"" heavy chain, because domain 3 retained all the thrombin inhibitory activity of the whole protein (Jiang et al., J. Biol. Chem. 267, 3712 (1992)). The thrombin inhibitory activity of the kininogens was later found to be associated with domain 4, the bradykinin sequence,: which was attached to the carboxyterminal end of isolated domain 3 prepared by proteolytic cleavage of whole LK (Hasan et al., Circulation 94, 517-528 (1996); Tayeh et al., J. Biol. Chem. 269, 16318-16325 (1994)). The thrombin inhibitory region of domain 4, the bradykinin sequence, demonstrated a number of features. This sequence did not prevent thrombin from binding to platelets and it did not prevent the thrombin receptor activation peptide (TRAP), SFLLRN (SEQ ID NO: 46), from stimulating calcium mobilization and platelet aggregation in platelets. This sequence from domain 4 prevented thrombin-activated platelets from losing an epitope to monoclonal antibody SPAN12. Monoclonal antibody SPAN12 is directed to the thrombin cleavage site on protease activated receptor 1 (PAR1) (Hasan et al., supra; Vu et al., Cell 64, 1057-1068 (1991); Brass et al., J. Biol. Chem. 267, 13795-13798 (1992)). Monoclonal antibody SPAN12 was raised to the peptide NATLDPRSFLLR (Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg, SEQ ID NO: 41) (Brass et. al., supra.). Further, bradykinin analog peptides prevented xcex1-thrombin from cleaving the peptide, NATLDPRSFLLR (Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg, SEQ ID NO: 41), between arginine and serine, the identical place on PAR1 that thrombin cleaves to activate this receptor. Although there are a number of peptide analogs of bradykinin that demonstrated thrombin inhibiting activity against platelet activation, the minimal sequences retaining this activity are the peptides, RPPGF (Arg-Pro-Pro-Gly-Phe, SEQ ID NO: 39), RPPG (Arg-Pro-Pro-Gly, SEQ ID NO: 45), and RPP (Arg-Pro-Pro). More recent investigations indicated that FITC-labeled (fluorescein isothiocyanate RPPGF (Arg-Pro-Pro-Gly-Phe, SEQ ID NO: 39) has the ability to directly bind to platelets (Hasan et al., Thromb Haemost. In Press, (1999)). These data indicated that the RPPGF (SEQ ID NO: 39) and related bradykinin analog peptides have the ability to bind to platelets to prevent thrombin-induced platelet activation.
The present invention relates to inhibition of thrombin-induced activation in human cells. Inhibition of thrombin activation of platelets can be either through an inhibitor of thrombin directed to the thrombin molecule itself or an inhibitor directed to substrates of thrombin. Protease activated receptor 1 (PAR1) is a specific substrate of thrombin to which this class of inhibitor is directed. The present invention is directed to inhibition of this thrombin substrate on any cell that expresses PAR1. These cells include platelets, endothelial cells, smooth muscle cells, fibroblasts, neuronal cells, or any other cell that contains this receptor. The present invention does not address inhibition of ADP-induced platelet activation as related to the thienopyridines class of agents, ticlopidine and clopidogrel. Similarly, the present invention does not address inhibition of platelet aggregation to the formation of the heterodimeric complex of platelet glycoprotein IIb/IIIa (i.e. integrin xcex1IIbxcex23). These compounds include the human-mouse chimeric monoclonal antibody 7E3c (ReoPro, abciximab), eptifibatide (Integrilin), and tirofiban (Aggrastat). This invention does not address aspirin inhibition of platelet activation by inhibition of platelet cyclooxygenase.
The following abbreviations have been used:
BK: bradykinin (SEQ ID NO: 40);
D3: domain 3 of kininogens;
D4: domain 4 of kininogen that is the bradykinin region;
FITC: fluorescein isothiocyanate;
HK: high molecular weight kininogen;
LK: low molecular weight kininogen;
MAP4RPPGF: A four-branched peptide consisting of a xcex2-alanine core with a single lysine attached at its amino terminal end followed by two additional lysines. Each lysine will then have two RPPGF (Arg-Pro-Pro-Gly-Phe, SEQ ID NO: 39) peptides attached by the phenylalanine to each of the lysines;
NAT12: peptide sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID NO: 41) that spans the ct-thrombin cleavage site on the thrombin receptor;
PAR1: protease activated receptor 1;
PTCA: percutaneous transluminal coronary angioplasty;
RPPGF: Arg-Pro-Pro-Gly-Phe (SEQ ID NO: 39);
RPPG: Arg-Pro-Pro-Gly (SEQ ID NO: 45);
RPP: Arg-Pro-Pro;
SPAN12: a monoclonal antibody specific for the sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID NO: 41) that spans the xcex1-thrombin cleavage site on the thrombin receptor; and
Z: nomenclature for any naturally occurring amino acid.
The invention relates to a series of compounds to inhibit thrombin-induced platelet or human cell activation upon administering an effective amount of a peptide that inhibits thrombin activation of platelets or human cells, wherein said peptide has an amino acid sequence of the formula:
Arg-Gly-Lys-Z4-Cys (SEQ ID NO: 43)xe2x80x83xe2x80x83(I)
wherein:
Z4 is any naturally occurring amino acid, excluding cysteine.
The invention also comprises a series of compounds to inhibit thrombin-induced platelet or human cell activation upon administering an effective amount of a peptide that inhibits thrombin activation of platelets or human cells, wherein said peptide has an amino acid sequence of the formula:
Arg-Gly-Asp-Z4-Cys (SEQ ID NO: 44)xe2x80x83xe2x80x83(II)
wherein:
Z4 is any naturally occurring amino acid, excluding cysteine.
The twenty amino acids (followed by their three letter abbreviations and one letter symbol) are set forth in TABLE VII below.
One embodiment of the invention comprises treating platelets or human cells with a compound of Formula I or Formula II to inhibit thrombin activation of platelets or activation of other cells, which express the thrombin receptor. Some of the preferred analogs include RGKWC, RGKKC, RGKLC, RGKTC, RGKRC, RGDWC, RGDFC, RGDEC, and RGDNC (SEQ ID NO""s 5, 14, 1, 2, 3, 20, 21, 22 and 32, respectively).
An object of administration of these peptides of Formulae I and II to cells is to prevent thrombosis, i.e., an occlusion of a vessel due to formation of a platelet-rich, fibrin-rich or a mixed platelet-fibrin thrombus. Accordingly, the invention relates to the foregoing analogs, and to the contact of these analogs with platelets and human cells which express the thrombin receptor to prevent thrombosis.